Review of possible modulation-dependent biological effects of radiofrequency fields

The biological effects of modulated radiofrequency (RF) electromagnetic fields have been a subject of debate since early publications more than 30 years ago, suggesting that relatively weak amplitude-modulated RF electromagnetic fields have specific biological effects different from the well-known thermal effects of RF energy. This discussion has been recently activated by the increasing human exposure to RF fields from wireless communication systems. Modulation is used in all wireless communication systems to enable the signal to carry information. A previous review in 1998 indicated that experimental evidence for modulation-specific effects of RF energy is weak. This article reviews recent studies (published after 1998) on the biological effects of modulated RF fields. The focus is on studies that have compared the effects of modulated and unmodulated (continuous wave) RF fields, or compared the effects of different kinds of modulations; studies that used only one type of signal are not included. While the majority of recent studies have reported no modulation-specific effects, there are a few interesting exceptions indicating that there may be specific effects from amplitude-modulated RF fields on the human central nervous system. These findings warrant follow-up studies.     

Analysis of the structure of magnetic fields that induced inhibition of stimulated neurite outgrowth

The important experiments showing nonlinear amplitude dependences of the neurite outgrowth in pheochromocytoma nerve cells due to ELF magnetic field exposure had been carried out in a nonuniform ac magnetic field. The nonuniformity entailed larger than expected variances in magnetic field magnitudes associated with specific levels of biological effects, thereby evoking a question about validity of the interpretations formulated for the case of a uniform field. In this work, we calculate the relative value of nonuniformity and deviations in ac magnetic field. It is shown that these factors do not affect the main conclusion in the original papers about the form of the amplitude dependence of the observed biological effect.     

Acute exposure to low-level CW and GSM-modulated 900 MHz radiofrequency does not affect Ba 2+ currents through voltage-gated calcium channels in rat cortical neurons

We have studied the non-thermal effects of radiofrequency (RF) electromagnetic fields (EMFs) on Ba(2+) currents (I Ba 2+) through voltage-gated calcium channels (VGCC), recorded in primary cultures of rat cortical neurons using the patch-clamp technique. To assess whether low-level acute RF field exposure could modify the amplitude and/or the voltage-dependence of I Ba 2+, Petri dishes containing cultured neurons were exposed for 1-3 periods of 90 s to 900 MHz RF-EMF continuous wave (CW) or amplitude-modulated according to global system mobile communication standard (GSM) during whole-cell recording. The specific absorption rates (SARs) were 2 W/kg for CW and 2 W/kg (time average value) for GSM-modulated signals, respectively. The results obtained indicate that single or multiple acute exposures to either CW or GSM-modulated 900 MHz RF-EMFs do not significantly alter the current amplitude or the current-voltage relationship of I Ba 2+, through VGCC.     

In vitro study of the effects of ELF electric fields on gene expression in human epidermal cells

An acceleration of differentiation, at the expense of proliferation, is observed after exposure of various biological models to low frequency and low amplitude electric and electromagnetic fields. Following these results showing significant modifications, we try to identify the biological mechanism involved at the cell level through microarray screening. For this study, we use epidermis cultures harvested from human abdominoplasty. Two platinum electrodes are used to apply the electric signal. The gene expressions of 38,500 well‐characterized human genes are analyzed using Affymetrix® microarray U133 Plus 2.0 chips. The protocol is repeated on three different patients. After three periods of exposure, a total of 24 chips have been processed. After the application of ELF electric fields, the microarray analysis confirms a modification of the gene expression of epidermis cells. Particularly, four up‐regulated genes (DKK1, TXNRD1, ATF3, and MME) and one down‐regulated gene (MACF1) are involved in the regulation of proliferation and differentiation. Expression of these five genes was also confirmed by real‐time rtPCR in all samples used for microarray analysis. These results corroborate an acceleration of cell differentiation at the expense of cell proliferation. Bioelectromagnetics 32:28–36, 2011. © 2010 Wiley‐Liss, Inc.     

In vitro effects of low frequency electromagnetic fields on osteoblast proliferation and maturation in an inflammatory environment

An in vitro model was set up to investigate the effects of low frequency pulsed electromagnetic fields (PEMF) and its induced electric fields on osteoblast cells under inflammatory conditions. Osteoblasts (7F2) were seeded on top of chitosan scaffolds and co-cultured with macrophage cells (RAW 264.7) growing on the bottom of culture wells, stimulated by lipopolysaccharide to release reactive oxygen species including nitric oxide (NO). The co-culture was exposed to PEMF (magnitude of the magnetic field = 1.5 mT; induced electric voltage = 2.5 mV; frequency = 75 Hz; pulse duration = 1.3 ms) for 9 h. The osteoblasts were examined for their proliferation, viability, alkaline phosphatase (ALP) activity, and genetic expressions of type I collagen (COL I) and osteocalcin (OC), immediately and 7 days after PEMF exposure (days 0 and 7). Macrophage cell viability and NO concentration in the medium were monitored before and after PEMF exposure. The PEMF-exposed co-culture released a significantly higher amount of NO (65 μM) compared to control (17 μM) on day 7. Despite the high level of NO in the medium that was reported to be cytotoxic, PEMF-exposed osteoblasts had enhanced cell proliferation (23%), viability (36%), and COL I mRNA expression (3.4-fold) compared to the controls. The osteoblasts subjected to the PEMF had 41% less ALP activity than the control, which was associated with the active cell proliferation and COL I expression. The expression of OC mRNA was not seen in either the PEMF or control group, indicating cells had not entered the mineralization stage by day 7.     

GSM base stations: short-term effects on well-being

The purpose of this study was to examine the effects of short-term GSM (Global System for Mobile Communications) cellular phone base station RF-EMF (radiofrequency electromagnetic fields) exposure on psychological symptoms (good mood, alertness, calmness) as measured by a standardized well-being questionnaire. Fifty-seven participants were selected and randomly assigned to one of three different exposure scenarios. Each of those scenarios subjected participants to five 50-min exposure sessions, with only the first four relevant for the study of psychological symptoms. Three exposure levels were created by shielding devices in a field laboratory, which could be installed or removed during the breaks between sessions such that double-blinded conditions prevailed. The overall median power flux densities were 5.2 microW/m(2) during "low," 153.6 microW/m(2) during "medium," and 2126.8 microW/m(2) during "high" exposure sessions. For scenario HM and MH, the first and third sessions were "low" exposure. The second session was "high" and the fourth was "medium" in scenario HM; and vice versa for scenario MH. Scenario LL had four successive "low" exposure sessions constituting the reference condition. Participants in scenarios HM and MH (high and medium exposure) were significantly calmer during those sessions than participants in scenario LL (low exposure throughout) (P = 0.042). However, no significant differences between exposure scenarios in the "good mood" or "alertness" factors were obtained. We conclude that short-term exposure to GSM base station signals may have an impact on well-being by reducing psychological arousal.     

Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells

Effects of magnetic fields (MFs) on cancer cells may depend on cell type and exposure conditions. Gene expression levels are different among cancer cells. However, the effect of MFs on cancer cells with different gene expressions is still unclear. In this study, the cancer cell lines BGC-823, MKN-45, MKN-28, A549, SPC-A1, and LOVO were exposed to a low-frequency MF. Specific parameters of MFs were determined. Furthermore, the potential of the MF to influence cancer cell growth with midkine (MK) expression was evaluated. Cell proliferation and cell cycle were detected using the CCK-8 assay and flow cytometry. Cell ultrastructure was observed by transmission electron microscopy. BGC-823 cells with over-expression of MK (BGC-MK cells) and stanniocalcin-1 were generated by plasmid construction and transfection. Results showed that exposure to a 0.4-T, 7.5 Hz MF inhibited the proliferation of BGC-823, MKN-28, A549, and LOVO cells, but not MKN-45 and SPC-A1 cells. Moreover, the inhibitory effect of the MF on BGC-MK cells was lower (12.3%) than that of BGC-823 cells (20.3%). Analysis of the cell cycle showed that exposure to the MF led to a significant increase in the S phase in BGC-823 cells, but not in BGC-MK cells. In addition, organelle morphology was modified in BGC-823 cells exposed to the MF. These results suggest that exposure to a 0.4-T, 7.5 Hz MF could inhibit tumor cell proliferation and disturb the cell cycle. The alteration of MK expression in cancer cells may be related to the inhibitory effect of the MF on these cells.     

Functionality of natural killer cells from end-stage cancer patients exposed to coherent electromagnetic fields

The main objective of our study is to investigate whether an enhancement of the immune system in end-stage cancer patients is achieved by exposure to coherent electromagnetic fields. For this reason, 15 end-stage cancer patients were exposed at low intensity, coherent electromagnetic fields at radiofrequencies ranging from 600 kHz–729 Hz, for 8 h/day, 6 days/week for 4 weeks. NKs number and cytotoxicity of NK T-lymphocytes versus K562 cancer cell line were estimated by flow cytometry, before and after exposure. Data showed that the exposure of the end-stage cancer patients to the coherent electromagnetic fields resulted in a significant increase of the number and the cytotoxicity of the NK T-lymphocytes against cancer cells, in all patients. Exposure to coherent EMFs at radiofrequencies increases the number and cytotoxicity of NK T-lymphocytes, which may contribute to the improvement of cancer patients' status.     

Extremely low frequency electromagnetic fields (ELF-EMFs) induce in vitro angiogenesis process in human endothelial cells

Effects of extremely low frequency (ELF) electromagnetic fields (EMFs) on activation of angiogenesis were analysed using cultured umbilical human vein endothelial cells (HUVECs). The cultures were exposed to a sinusoidal EMF to intensity of 1 mT, 50 Hz for up to 12 h. EMFs increased the degree of endothelial cell proliferation and tubule formation, coupled by an acceleration in the process of wound healing. Since this process is physiologically accompanied by a large modification in the structural organization of actin and focal adhesions, we analyzed the rearrangement of some cytoskeleton elements demonstrating a major reorganization of the fibres and of the focal adhesion complexes after EMF exposure. Finally, Western blot analysis revealed a significant increase in phosphorylation as well as the overall expression of VEGF receptor 2 (KDR/Flk-1) suggesting that EMFs may modulate in vitro some endothelial functions correlated to angiogenesis through signal transduction pathways dependent on VEGF.     

Electromagnetic and thermal characterization of an UHF-applicator for concurrent irradiation and high resolution non-perturbing optical microscopy of cells

To permit trans-illuminated, high-resolution optical microscopy during unperturbed ultrahigh frequency (UHF) irradiation, a novel new class of applicator has been designed based upon a shielded-pair transmission line. As constructed and tested with water-immersion optics and air cooling, the applicator works most robustly over 700-1100 MHz and permits SARs at the cell layer as high as 50 W/kg before the steady state temperature rise at the cell-layer exceeds 0.5 K.     

Complex effects of long-term 50 Hz magnetic field exposure in vivo on immune functions in female Sprague-Dawley rats depend on duration of exposure

In previous studies we have demonstrated that 50 Hz, 100 μT magnetic field (MF) exposure of female Sprague-Dawley rats for 13 weeks significantly enhances the development and growth of mammary tumors in a breast cancer model. The present study was designed to test the hypothesis that, at least in part, the tumor (co)promoting effect of MF exposure is due to MF effects on the immune surveillance system, which is of critical importance in protecting an organism against the development and growth of tumors. For this purpose, female Sprague-Dawley rats of the same age as in the mammary tumor experiments were continuously exposed for different periods (2, 4, 8, and 13 weeks) to a 50 Hz, 100 μT MF. Control groups were sham-exposed simultaneously. Following the different exposure periods, splenic lymphocytes were cultured and the proliferative responses to the T-cell-selective mitogen concanavalin A (Con A) and the B-cell-selective pokeweed mitogen (PWM) were determined. Furthermore, the production of interleukin-1 (IL-1) was determined in the splenocyte cultures. The mitogenic responsiveness of T cells was markedly enhanced after 2 weeks of MF exposure, suggesting a co-mitogenic action of MF. A significant, but less marked increase in T-cell mitogenesis was seen after 4 weeks of MF exposure, whereas no difference from sham controls was determined after 8 weeks, indicating adaptation or tolerance to this effect of MF exposure. Following 13 weeks of MF exposure, a significant decrease in the mitogenic responsiveness of lymphocytes to Con A was obtained. This triphasic alteration in T-cell function (i.e., activation, tolerance, and suppression) during prolonged MF exposure resembles alterations observed during chronic administration of mild stressors, substantiating the hypothesis that cells respond to MF in the same way as they do to other environmental stresses. In contrast to T cells, the mitogenic responsiveness of B cells and IL-1 production of PWM-stimulated cells were not altered during MF exposure. The data demonstrate that MF in vivo exposure of female rats induces complex effects on the mitogenic responsiveness of T cells, which may lead to impaired immune surveillance after long-term exposure. Bioelectromagnetics 19:259–270, 1998. © 1998 Wiley-Liss, Inc.     

Non-thermal effects of electromagnetic fields at mobile phone frequency on the refolding of an intracellular protein: myoglobin

Non-thermal effects induced by exposure to microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, have been observed on the refolding kinetics of the heme binding site in an intracellular protein: tuna myoglobin, starting from acidic conditions. We have selected myoglobin because it can be considered a good model to study protein interactions with MW-EMF for its well-known high-resolution crystallographic structure. Myoglobin solutions at pH 3.0 were subjected to 3 h exposure to microwave field (with a specific absorption rate of 51 +/- 1 mW/g); the heme site refolding has been followed by measuring the molecular absorption in the Soret spectral region and the data were fitted to a bi-exponential model. The kinetics of exposed samples appear to be slowered by MW-EMF action. Moreover, the tryptophanyl lifetime distribution of the exposed protein, as deduced by the analysis of the fluorescence emission decay from its single tryptophan, appears sharper if compared to non-exposed protein samples. This observation suggests that the presence of MW-EMF could affect the propensity of protein molecules to populate specific conformational substates among which myoglobin molecules fluctuate at acidic pH. Changes in the structural fluctuation caused by MW perturbation can affect differently the aggregation process that occurs competitively during the protein folding, so representing a potential risk for protein "misfolding." These data suggest that MW-EMF could have also biochemical and, consequently, biological effects on eukaryotic cells that are still under investigation.     

Non-thermal effects in the microwave induced unfolding of proteins observed by chaperone binding

We study the effect of microwaves at 2,450 MHz on protein unfolding using surface plasmon resonance sensing. Our experimental method makes use of the fact that unfolding proteins tend to bind to chaperones on their unfolding pathway and this attachment is readily monitored by surface plasmon resonance. We use the protein citrate synthase (CS) for this study as it shows strong binding to the chaperone alpha crystallin when stressed by exposure to excess temperature. The results of microwave heating are compared with the effect of ambient heating and a combination of ambient and microwave heating to the same final temperature. We study the temperature distributions during the heating process. We show that microwaves cause a significantly higher degree of unfolding than conventional thermal stress for protein solutions heated to the same maximum temperature.     

Acetylsalicylic acid has no effects on various isolated immune cells in vitro

In addition to the well known biological effects of acetylsalicylic acid (ASA), its stimulating effect on the immune system has recently been described. In the present study, therefore, the influence of ASA on isolated leucocytes was investigated in vitro. Various concentrations of ASA, representing therapeutic concentrations, had neither an inhibitory nor a stimulating influence on the cytotoxicity of C1316-positive cells. The phytohaemagglutinin-induced proliferation of lymphocytes and the phagocytosis activity of monocytes and neutrophilic granulocytes was similarly unaffected. These results would indicate that the immunostimulating effects already described for ASA cannot be examined in isolated leucocytes in vitro and should be attributed to an interaction of various effector cells in vivo.     

A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis Elegans

We have previously reported that low intensity microwave exposure (0.75-1.0 GHz CW at 0.5 W; SAR 4-40 mW/kg) can induce an apparently non-thermal heat-shock response in Caenorhabditis elegans worms carrying hsp16-1::reporter genes. Using matched copper TEM cells for both sham and exposed groups, we can detect only modest reporter induction in the latter exposed group (15-20% after 2.5 h at 26 degrees C, rising to approximately 50% after 20 h). Traceable calibration of our copper TEM cell by the National Physical Laboratory (NPL) reveals significant power loss within the cell (8.5% at 1.0 GHz), accompanied by slight heating of exposed samples (approximately 0.3 degrees C at 1.0 W). Thus, exposed samples are in fact slightly warmer (by < or =0.2 degrees C at 0.5 W) than sham controls. Following NPL recommendations, our TEM cell design was modified with the aim of reducing both power loss and consequent heating. In the modified silver-plated cell, power loss is only 1.5% at 1.0 GHz, and sample warming is reduced to approximately 0.15 degrees C at 1.0 W (i.e., < or =0.1 degrees C at 0.5 W). Under sham:sham conditions, there is no difference in reporter expression between the modified silver-plated TEM cell and an unmodified copper cell. However, worms exposed to microwaves (1.0 GHz and 0.5 W) in the silver-plated cell also show no detectable induction of reporter expression relative to sham controls in the copper cell. Thus, the 20% "microwave induction" observed using two copper cells may be caused by a small temperature difference between sham and exposed conditions. In worms incubated for 2.5 h at 26.0, 26.2, and 27.0 degrees C with no microwave field, there is a consistent and significant increase in reporter expression between 26.0 and 26.2 degrees C (by approximately 20% in each of the six independent runs), but paradoxically expression levels at 27.0 degrees C are similar to those seen at 26.0 degrees C. This surprising result is in line with other evidence pointing towards complex regulation of hsp16-1 gene expression across the sub-heat-shock range of 25-27.5 degrees C in C. elegans. We conclude that our original interpretation of a non-thermal effect of microwaves cannot be sustained; at least part of the explanation appears to be thermal.     

In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2–0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed that the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate induces hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate that a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage in an oxidatively stressed erythrocyte system. In fact, exposure of intact erythrocytes incubated with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents. Bioelectromagnetics 18:125–131, 1997. © 1997 Wiley-Liss, Inc.     

Combined effect of pulsed electromagnetic field and sound wave on In vitro and In vivo neural differentiation of human mesenchymal stem cells

Biophysical wave stimulus has been used as an effective tool to promote cellular maturation and differentiation in the construction of engineered tissue. Pulsed electromagnetic fields (PEMFs) and sound waves have been selected as effective stimuli that can promote neural differentiation. The aim of this study was to investigate the synergistic effect of PEMFs and sound waves on the neural differentiation potential in vitro and in vivo using human bone marrow mesenchymal stem cells (hBM–MSCs). In vitro, neural‐related genes in hBM–MSCs were accelerated by the combined exposure to both waves more than by individual exposure to PEMFs or sound waves. The combined wave also up‐regulated the expression of neural and synaptic‐related proteins in a three‐dimensional (3‐D) culture system through the phosphorylation of extracellular signal‐related kinase. In a mouse model of photochemically induced ischemia, exposure to the combined wave reduced the infarction volume and improved post‐injury behavioral activity. These results indicate that a combined stimulus of biophysical waves, PEMFs and sound can enhance and possibly affect the differentiation of MSCs into neural cells. Our study is meaningful for highlighting the potential of combined wave for neurogenic effects and providing new therapeutic approaches for neural cell therapy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:201–211, 2017